Abstract

Non-small cell lung cancer (NSCLC) patients account for over 80% of all lung cancer cases and have a poor outcome with current radiotherapy regimens. NSCLC has been the subject of many studies characterizing mechanisms of initiation, proliferation, invasion and treatment response. Recent trends in radiotherapy show an increase in the use of ablative radiotherapy (ART) in inoperable small size tumors. Clinical studies have shown response rates up to 90% in NSCLC patients treated with ART compared to 15-20% in patients treated with conventional fractionated radiotherapy (FRT). However, the biological determinants of this response have not been investigated and recent analysis of patterns of failure in patients treated with ART show an increased tendency towards distant metastatic recurrence.

We investigated the biological response of NSCLC cell lines with different molecular subtypes including, A549, H1975 and HCC827 to ART and FRT. Radiation doses of 8Gy and 12Gy were delivered in multiple fractions or single fraction. The response to radiation was investigated using several cellular assays including clonogenic survival, cell proliferation, matrigel invasion, wound-healing, morphological characterization and senescence-associated beta-galactosidase.

ART significantly reduced cell proliferation and clonogenic survival compared to FRT in A549 cells. In addition, a significant increase in the number of senescent cells as well as large, polynucleated cells was observed in the ART-treated group compared to the FRT-treated group. This differential response to delivery approach (ART vs FRT) was not observed in HCC827 or H1975 cells which harbor EGFR mutations. Both ART and FRT inhibited cell proliferation and clonogenic survival to similar levels in HCC827 and H1975 cells.

In contrast to reduced cell proliferation and clonogenicity, ART significantly increased the invasive phenotype of A549 cells, but not HCC827 or H1975 cells compared to FRT. Western blot analysis of several markers of invasion, including cMET, AKT, ERK, SPARC and FAK revealed a significant down regulation of SPARC in A549 cells exposed to ART, but not FRT. Further analysis of cells in Boyden chambers showed that ART-induced senescent cells are capable of migration/invasion.

Our results unequivocally demonstrate that response to ART is cell-line dependent. A549 cells, which harbor wild-type EGFR have a differential response to radiotherapy based on delivery approach. Furthermore, A549 cell exposed to ART have significantly increased invasive and migratory capacity compared to FRT. Our findings suggest that the extracellular matrix glycoprotein, SPARC is involved in the modulation of radiation-induced invasion/migration. These findings can have significant implications for NSCLC patients undergoing ART and underscore the importance of understanding the underlying biology for effective disease management.